Application of deubiquitinase USP8 inhibitors in the preparation of drugs for treating Japanese encephalitis

Abstract

This invention relates to the field of pharmaceutical technology, specifically the application of deubiquitinase USP8 inhibitors in the preparation of drugs for treating Japanese encephalitis. This invention uses human neuroblastoma cells (SH-SY5Y) as target cells and utilizes inhibitors from a library of ubiquitination compounds to target and inhibit key enzymes in the ubiquitination pathway, aiming to identify host factors associated with Japanese encephalitis virus (JEV) infection of SH-SY5Y cells. This helps to understand the mechanism by which JEV invades the central nervous system and causes neuronal cell damage, and also provides new targets for the treatment of JEV-induced central nervous system infections and pathogenesis. This invention experimentally demonstrates that deubiquitinase USP8 inhibitors have the property of inhibiting JEV infection of SH-SY5Y cells. This invention provides the application of deubiquitinase USP8 inhibitors in the preparation of drugs for treating Japanese encephalitis, offering new targets and treatment strategies for the prevention and treatment of JEV.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical technology, specifically the application of deubiquitinase USP8 inhibitors in the preparation of drugs for treating Japanese encephalitis. Background Technology

[0002] Japanese encephalitis (JE) is caused by the Japanese encephalitis virus (JEV), which is transmitted by mosquitoes. JEV belongs to the Flaviviridae family and the Flavivirus genus and is a single-stranded positive-sense RNA virus. JEV is neuropathogenic and can cause Japanese encephalitis, which is characterized by inflammation of the brain parenchyma. Severe Japanese encephalitis patients mainly present with symptoms such as high fever, headache, and coma. The mortality rate of those with disease symptoms can be as high as 30%, and about 30%-50% of severe survivors have neurological sequelae such as paralysis and intellectual disability (Ashraf U, Ding Z, Deng S, et al. Pathogenicity and virulence of Japanese encephalitis virus: Neuroinflammation and neuronal cell damage. Virulence. 2021; 12(1):968-980.). Japanese encephalitis is mainly prevalent in East Asia, Southeast Asia, and parts of Oceania. The main control measures are vaccination and mosquito elimination. Although vaccination has significantly reduced the incidence of Japanese encephalitis (JE), the incidence continues to rise. In recent years, the development of anti-JE drugs has been a hot topic in medical and biological research both domestically and internationally. Through the tireless efforts of researchers, several drugs with anti-JE activity have been discovered. These drugs can be broadly classified into two categories based on their properties: first, synthetic antiviral drugs, including nucleic acid-targeted therapeutic agents, nucleic acid analogs, cytokines, flavonoids, and antibiotics; and second, antiviral drugs obtained from natural medicines, including phenolic compounds, arctiinogen, and extracts of some traditional Chinese medicine components. However, the evaluation of the efficacy of these drugs is mainly limited to animal models, cellular, and molecular levels, and further research is needed to determine whether they can be applied clinically.

[0003] Ubiquitin-specific protease 8 (USP8) is a cysteine ​​enzyme belonging to the deubiquitinating enzyme (DUB) superfamily. It is approximately 130 kDa in size. DUB family proteins are proteases that specifically hydrolyze isopeptide bonds between ubiquitin and target proteins or between ubiquitin molecules; therefore, USP8 is also known as ubiquitin isopeptidase Y (UBPY). Like many other DUBs, USP8 is characterized by its multi-domain structure. In addition to its catalytic domain, USP8 also possesses specific protein-protein interaction domains. These domains facilitate the recruitment of USP8 substrates and the regulation and targeting of different protein complexes (Dufner A, Knobeloch KP. Ubiquitin-specific protease 8 (USP8 / UBPy): a prototypic multidomain deubiquitinating enzyme with pleiotropic functions. Biochem Soc Trans. 2019; 47(6):1867-1879.). Studies have shown that USP8 plays multiple roles in living organisms, and abnormal USP8 function is closely related to various diseases, including cancer and neurodegenerative diseases. USP8 can also directly regulate the degradation of epidermal growth factor receptor (EGFR). Recent studies have also found that USP8 is involved in the infection and pathogenesis of various viruses. For example, Zika virus can infect cells by using the function of USP8 (Zheng Y, Liu Q, Wu Y, et al. Zika virus elicitsinflammation to evade antiviral response by cleaving cGAS via NS1-caspase-1axis. EMBO J. 2018; 37(18):e99347.).

[0004] (E)-9-(ethoxyimino)-9H-indeno[1,2-b]pyrazine-2,3-dicarbonitrile, also known as deubiquitinase inhibitor 2 (DUB-IN-2), is an indeno[1,2-b]pyrazine compound (chemical structure shown in Formula I) that targets and inhibits the activity of USP8. USP8 is often overactivated in tumor cells, and activated USP8 participates in regulating downstream signal transduction pathways, which is closely related to the malignant behavior of tumor cells. Studies have shown that the mutation rate of USP8 in pituitary adrenocorticotropic hormone adenomas is 35%-60%. DUB-IN-2 has been shown to function in mouse pituitary tumor cells (AtT-20) (Kageyama K, Asari Y, Sugimoto Y, et al. Ubiquitin-specific protease 8 inhibitor suppresses adrenocorticotropic hormone production and corticotroph tumor cell proliferation. Endocr J. 2020; 67(2):177-184.). Xiong et al. reported that by using DUB-IN-2, the inflamed tumor microenvironment (TME) can be reshaped, significantly enhancing the efficacy of anti-PD-1 / PD-L1 immunotherapy (Xiong W, Gao X, Zhang T, et al. USP8 inhibition reshapes an inflamed tumor microenvironment that potentiates the immunotherapy. Nat Commun. 2022; 13(1):1700.).

[0005] However, there are currently no reports on the therapeutic effects of USP8 inhibitors in Japanese encephalitis. Summary of the Invention

[0006] The purpose of this invention is to provide the application of deubiquitinase USP8 inhibitors in the preparation of drugs for treating Japanese encephalitis.

[0007] A first aspect of the present invention provides the use of a deubiquitinase USP8 inhibitor in the preparation of a medicament for treating Japanese encephalitis.

[0008] Furthermore, the aforementioned deubiquitinase USP8 inhibitor is (E)-9-(Ethoxyimino)-9H-indeno[1,2-b]pyrazine-2,3-dicarbonitrile, also known as deubiquitinase inhibitor 2 (DUB-IN-2), which is an indeno[1,2-b]pyrazine compound (structural formula shown in Formula I) capable of targeting and inhibiting the activity of USP8.

[0009]

[0010] Furthermore, the use of the aforementioned deubiquitinase USP8 inhibitor in the preparation of drugs that inhibit Japanese encephalitis virus infection and cytotoxicity.

[0011] A second aspect of the present invention provides a medicament for treating Japanese encephalitis, comprising:

[0012] (A) An effective amount of a deubiquitinase USP8 inhibitor; and

[0013] (B) Pharmaceutically or immunologically acceptable carriers or excipients.

[0014] Furthermore, the aforementioned deubiquitinase USP8 inhibitor is deubiquitinase inhibitor 2 (DUB-IN-2).

[0015] The advantages of this invention are:

[0016] 1. This invention uses human neuroblastoma cells (SH-SY5Y) as target cells and utilizes inhibitors from a library of ubiquitination compounds to target and inhibit key enzymes in the ubiquitination pathway in order to identify host factors associated with Japanese encephalitis virus (JEV) infection of SH-SY5Y cells. This helps to understand the mechanism by which JEV invades the central nervous system and causes neuronal cell damage, and also provides new targets for therapeutic drugs targeting JEV-induced central nervous system infections and pathogenesis. This invention experimentally discovered that the deubiquitinating enzyme USP8 inhibitor also has the property of inhibiting Japanese encephalitis virus infection of SH-SY5Y cells.

[0017] 2. This invention provides the application of deubiquitinase USP8 inhibitor in the preparation of drugs for treating Japanese encephalitis, providing a new drug for the prevention and treatment of Japanese encephalitis virus, with good market value and clinical application prospects. Attached Figure Description

[0018] Figure 1Figure A shows partial immunofluorescence detection results of screening a library of ubiquitinated compounds using the JEV in vitro cell culture system, while Figure B shows the statistical graph of the corresponding viral infection inhibition rate.

[0019] Control: JEV-infected SH-SY5Y cells without any inhibitors (blank control group);

[0020] Chloroquine: JEV infection of SH-SY5Y cells with 100 μM chloroquine (positive control group);

[0021] Inhibitor: JEVs were added at a concentration of 5 μM to target different key enzymes in the ubiquitin-proteasome system and infected SH-SY5Y cells (experimental group).

[0022] Figure 2 This is a graph showing the inhibitory effect of the USP8 inhibitor DUB-IN-2 on viral infection. In the graph, A is a schematic diagram of the inhibition of JEV infection and cytotoxicity detection after using different concentrations of DUB-IN-2 on target cells. The main vertical axis represents the amount of JEV virus, and the secondary vertical axis represents the effect on cytotoxicity. B is an immunofluorescence detection graph showing the effect of different concentrations of DUB-IN-2 on viral infectivity after acting on target cells. Detailed Implementation

[0023] The specific implementation methods provided by the present invention will be described in detail below with reference to the embodiments.

[0024] Example 1

[0025] I. Experimental Materials

[0026] Ubiquitination Compound Library (Cat. No.: HY-L050), purchased from MCE.

[0027] DUB-IN-2 is a ubiquitin-specific protease 8 inhibitor (Cat. No.: HY-50737A), purchased from MCE.

[0028] The human neuroblastoma strain SH-SY5Y was purchased from ATCC, accession number: ATCC CRL-2266.

[0029] II. Experimental Methods

[0030] 1. siRNA interference

[0031] 1.1 RNA transfection

[0032] The transfection procedure is as per the Lipofectamine 2000 instruction manual.

[0033] 1) 12-16 hours in advance, seed SH-SY5Y cells (purchased from ATCC, accession number: ATCC CRL-2266) on 24-well cell culture plates to achieve a cell density of 80%-90% at the time of transfection.

[0034] 2) Add 2 μL of Lipofectamine 2000 to 50 μL of opti-MEM and mix gently. Incubate at room temperature for 5 min. Separately, mix 5 μL of 5 μM interfering RNA with 50 μL of opti-MEM. After incubation, add the diluted Lipofectamine 2000 transfection reagent to the diluted RNA and mix gently by pipetting. After incubating at room temperature for 20 min, add the RNA to SH-SY5Y cells and add 400 μL of opti-MEM to bring the final RNA concentration to 50 nM.

[0035] 3) Replace with fresh culture medium containing antibiotics 6-8 hours after transfection.

[0036] 2. Real-time quantitative PCR (RT-PCR) for detecting JEV viral load

[0037] 1) Total RNA was extracted from cells in the control and treatment groups using TRIzol. The specific steps are as follows:

[0038] After treating the target cells, discard the culture supernatant. Add 1 ml of TRIzol to the cells and mix thoroughly to lyse the cells at room temperature for 3-5 min. Add 1 / 5 volume of chloroform and mix vigorously manually for 15 s. Centrifuge at 12,000 rpm for 15 min at 4 °C. Collect the upper aqueous phase and transfer it to a new EP tube. Add an equal volume of isopropanol, mix thoroughly, and precipitate at room temperature for 10 min. Centrifuge at 12,000 rpm for 10 min at 4 °C. Discard the supernatant and add 1 ml of pre-chilled 75% ethanol. Centrifuge at 12,000 rpm for 5 min at 4 °C. Discard the supernatant completely, air-dry the RNA precipitate at room temperature, and dissolve the precipitate in DEPC-treated water to obtain total RNA.

[0039] 2) Obtain cDNA from control and interference group cells using the Takara reverse transcription kit. The specific steps are as follows:

[0040] Add the following reaction mixture to the PCR tube.

[0041]

[0042] Mix gently and thoroughly, react at 37°C for 15 min, then heat at 85°C for 5 s to inactivate reverse transcriptase.

[0043] 3) Quantitative Real-Time RT-PCR Detection

[0044] The reaction was carried out using Takara's SYBR Premix Ex Taq kit, and the reaction system is as follows.

[0045]

[0046]

[0047] Two-step amplification was performed using a Rotor Gene 3000A instrument: pre-denaturation at 95℃ for 2 min, followed by 40 PCR cycles of 95℃ for 5 s and 60℃ for 30 s.

[0048] 3. JEV virus infection of SH-SY5Y cells

[0049] 3.1 JEV virus infection experiment in SH-SY5Y cells

[0050] SH-SY5Y cells were transfected with RNA for 48 hours before JEV virus infection experiments were performed. The culture supernatant was aspirated, washed twice with pre-warmed PBS, and inoculated with JEV at an MOI of 0.5. After incubation at 37°C for 2 hours, the virus solution was discarded, and the cells were washed three times with pre-warmed PBS. Fresh culture medium was then added for further culture.

[0051] 3.2 Immunofluorescence staining to detect JEV antigen expression

[0052] After SH-SY5Y cells were infected with the virus, they were cultured for another 48 hours. The expression of viral antigens was detected by immunofluorescence. The specific steps are as follows:

[0053] 1) Cell fixation: Remove the culture medium from the 96-well plate, wash the cells twice with PBS, add 100 μl of pre-cooled methanol to each well, fix at -20℃ for 20 min, and wash the cells three times with pre-cooled PBS.

[0054] 2) Permeabilization: Add 100 μl of 0.1% Triton X-100 to each well of the fixed cells, incubate at room temperature for 15 min, and wash 3 times with pre-cooled PBS.

[0055] 3) Seal: Add 100 μl of 3% BSA to each well and incubate at room temperature for 1 h.

[0056] 4) Primary antibody incubation: Add 100 μl of JEV-specific rabbit monoclonal antibody GTX125868 (1:1000 dilution) to each well, incubate at room temperature for 1 h, and wash 3 times with pre-cooled PBS.

[0057] 5) Secondary antibody incubation: Add 100 μl of AF 488 fluorescently labeled anti-rabbit IgG (1:1000 dilution) to each well, incubate at room temperature in the dark for 1 h, and wash twice with pre-cooled PBS in the dark.

[0058] 6) Labeling cell nuclei: Add the nuclear fluorescent dye DAPI (1:10000, diluted with PBS) to each well, incubate at room temperature in the dark for 15 min, and wash 3 times with pre-cooled PBS in the dark.

[0059] 7) Detect and count the number of green AF 488 positive cell clones under a fluorescence microscope.

[0060] 4. Western blot for protein immunoblotting

[0061] 1) Total protein was extracted from SH-SY5Y cells in different treatment groups using protein lysis buffer.

[0062] 2) After protein quantification, 30 μg of protein was added to a 12.5% ​​polyacrylamide gel for electrophoresis, and the corresponding bands were transferred to a PVDF membrane using an electroporator.

[0063] 3) Block the non-specific sites of the protein with 5% skim milk, then block with USP8 or EGFR antibody, incubate overnight at 4°C, and wash three times with TBST buffer to remove the primary antibody.

[0064] 4) Then incubate with HRP-labeled secondary antibody at room temperature for 2 hours, followed by washing three times with TBST buffer.

[0065] 5) Finally, develop the color using a colorimetric solution and take photos for analysis.

[0066] 5. Screening of ubiquitinated compound libraries

[0067] 1) Grouping: The experiment was divided into a blank control group, a positive drug control group and an experimental group. Each drug in each group was set up with 3 replicates. The experiment was independently repeated three times.

[0068] Control: JEV-infected SH-SY5Y cells without any inhibitors (blank control group);

[0069] Chloroquine: JEV infection of SH-SY5Y cells with 100 μM chloroquine (positive control group);

[0070] Inhibitor: JEVs infected with JEVs targeting different key enzymes in the ubiquitin-proteasome system at a concentration of 5 μM (experimental group).

[0071] 2) 12-16 hours in advance, seed SH-SY5Y cells onto 96-well cell culture plates to achieve a cell density of 80%-90% at the time of treatment. Aspirate the culture supernatant, wash twice with PBS pre-warmed to 37°C, add 100 μl of different inhibitors to each well, and incubate at 37°C for 6 hours. The positive control group is given 100 μl of 100 μM chloroquine in whole culture medium, and the blank control group is given an equal volume of whole culture medium.

[0072] Discard the inhibitor solution, add an equal amount of JEV (MOI = 0.5) to each well, incubate at 37°C for 48 h, then discard the virus solution and wash three times with PBS. Immunofluorescence detection is performed using the same method as in step 3.2.

[0073] 6. Inhibitor (DUB-IN-2) action

[0074] 1) Grouping: The experiment was divided into a blank control group, a positive drug control group, and an inhibitor interference group. Each drug concentration in each group was set up with 3 replicates, and the experiment was independently repeated three times.

[0075] 2) 12-16 hours in advance, seed SH-SY5Y cells onto 24-well cell culture plates to achieve a cell density of 80%-90% at the time of treatment. Aspirate the culture supernatant, wash twice with PBS pre-warmed to 37°C, add 500 μl of different concentration gradients of the drug to each well, and incubate at 37°C for 6 hours. The positive control group was given 100 μM chloroquine in whole culture medium, and the blank control group was given an equal volume of whole culture medium.

[0076] Discard the chemical reagent solution, add an equal amount of JEV (MOI = 0.5) to each well, incubate at 37°C for 2 hours, then discard the virus solution. Wash three times with PBS, and add proteinase K solution (1 mg / ml) to remove viral particles bound to the surface of target cells. Subsequent culture and detection of JEV-infected target cells are performed in step 3.2.

[0077] 7. Cytotoxicity assay

[0078] The effect of DUB-IN-2 on cell proliferation was detected using the CCK-8 assay. The specific steps are as follows:

[0079] Logarithmic growth phase cells were collected and seeded at a density of 3000 cells per well in 96-well plates. After overnight cell attachment, DUB-IN-2 was added, and cell proliferation was assessed after 48 hours of culture. The original culture medium was discarded, and 110 μL of fresh medium containing 10 μL of CCK-8 was added to each well. After 3 hours of culture, the absorbance of each well was measured at 450 nm using a multi-mode microplate reader. The experiment was independently repeated three times, and the average value was calculated.

[0080] III. Experimental Results

[0081] Using the JEV in vitro cell culture system, screening of a library of ubiquitinated compounds revealed that the deubiquitination enzyme USP8 inhibitor DUB-IN-2 exhibited good anti-JEV activity, inhibiting JEV infection of SH-SY5Y cells. Figure 1 A). We also set up a blank control group (no drug added, Control), a positive drug control group (100 μM Chloroquine), and an inhibitor treatment group (5 μM). After 6 h of drug treatment, patients were infected with JEV for 48 h. Viral antigens were then detected by indirect immunofluorescence, and the infection rate was calculated and normalized to the Control group. Candidate drugs were included based on a relative inhibition rate of >50% against JEV (i.e., infection rate <50%, the dotted line in the figure represents infection rate = 50%) and a superior inhibitory effect compared to Chloroquine. Among these, the deubiquitinase USP8 inhibitor DUB-IN-2 showed a relatively significant inhibitory effect on JEV. Figure 1 B).

[0082] Furthermore, the effects on viral infection were examined after treatment with different concentration gradients of the deubiquitinating enzyme USP8 inhibitor DUB-IN-2. The results showed that as the inhibitor concentration increased, the viral load of JEV gradually decreased, while different concentrations of inhibitor had no significant effect on cell viability. Figure 2 A) The results obtained by indirect immunofluorescence assay for viral antigens were consistent. Figure 2 B) These results indicate that the deubiquitinase USP8 inhibitor DUB-IN-2 can inhibit JEV infection and that its antiviral activity is positively correlated with drug concentration.

[0083] To clarify the effect of the USP8 inhibitor, the USP8 deubiquitinase inhibitor DUB-IN-2, on JEV infection, USP8 protein expression was detected by Western blotting after transfection with USP8 siRNA. The results showed that transfection with USP8 siRNA significantly inhibited USP8 protein expression. Immunofluorescence assays of viral antigens indicated that downregulation of USP8 significantly reduced JEV infection of SH-SY5Y cells. These results suggest that, compared to control cells, downregulation of USP8 significantly reduced the infectivity of JEV in SH-SY5Y cells and decreased viral load. Therefore, the deubiquitinase USP8 could serve as a novel drug target for inhibiting JEV infection of SH-SY5Y cells.

[0084] The above experimental results demonstrate that the present invention found that the deubiquitinating enzyme USP8 inhibitor can significantly inhibit JEV infection in SH-SY5Y cells. Furthermore, it was found that inhibiting the expression of the deubiquitinating enzyme USP8 protein molecule can reduce JEV infection, thus exerting an antiviral effect. This invention provides a new therapeutic drug and target for the prevention and treatment of JEV-induced Japanese encephalitis, possessing significant market value and clinical application prospects.

[0085] The preferred embodiments of the present invention have been described in detail above, but the present invention is not limited to the embodiments described. Those skilled in the art can make various equivalent modifications or substitutions without departing from the spirit of the present invention, and these equivalent modifications or substitutions are all included within the scope defined by the claims of this application.

Claims

1. The application of a deubiquitinase USP8 inhibitor in the preparation of drugs for treating Japanese encephalitis, characterized in that, The aforementioned deubiquitinase USP8 inhibitor is (E)-9-(ethoxyimino)-9H-indeno[1,2-b]pyrazine-2,3-dicarboxynitrile, with the structural formula shown in Formula I: Formula I.

2. The use of the deubiquitinase USP8 inhibitor according to claim 1 in the preparation of a drug for treating Japanese encephalitis, characterized in that, The application of the aforementioned deubiquitinase USP8 inhibitor in the preparation of drugs that inhibit Japanese encephalitis virus infection and cytotoxicity.

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